Long-term objectives: Hepatitis C virus (HCV) is one of the most important causes of hepatocellular carcinoma. The long-term goal of this proposal is to understand the mechanism of hepatocarcinogenesis by hepatitis C virus, specifically focusing on the role of the viral core protein. Rationale: Our previous findings on HCV-infected Raji cells and HCV core protein-transgenic mice have suggested several pathways for HCV-induced hepatocarcinogenesis, including the possible role of inflammation and of the inhibition of DMAdamage repair by HCV proteins. Furthermore, the activation of toll-like receptor 4 (TLR4) by HCV NS5A protein suggested possible synergism between HCV and alcohol/diabetes since the latter induces enhanced sensitivity to endotoxin. These are novel mechanisms of viral oncogenesis by a cytoplasmic RNA virus. Specific aims: In this subproject, we will use both culture cell lines infected by HCV in vitro and HCV core protein-transgenic mice to explore the potential mechanisms of HCV oncogenesis. Three specific aims are proposed: 1) Establish the importance of inflammation in HCV oncogenesis. The effects of HCV infection on NFkB and IKKbeta in HCV infection will be studied in cultured cell lines and in IKKbeta-conditional knockout mice. Furthermore, transgenic mice expressing HCV proteins in B cells will be generated to examine the possible effect of inflammatory cytokines on oncogenesis. 2) Using NS5A-transgenic mice, which have enhanced TLR4 expression, we will study the possible synergism between HCV and alcohol/diabetes. Core/NS5A transgenic-TLR4-/- mice will be generated, and the tumor incidence induced by alcohol/obesity will be compared with the core/NS5A Tg mice to assess the role of TLR4. 3) Study the mechanism of inhibition of DMAdamage-repair by HCV. The DMAdamage sensor and response complexes will be studied in HCV-infected cells or cells expressing HCV core protein. Various DMA repair pathwayswill be studied. Finally proteomic analysis in HCV-infected and core- or NS3- expressing cells will be performed to identify other potential target proteins of HCV. These studies are expected to generate knowledge on the possible mechanism of HCV oncogenesis.